Cholesterol is an important component of eukaryotic cell membranes. It has been shown to modify diverse cellular processes from membrane transport events to transcriptional regulation in cells that underlie both neurodegenerative and heart diseases. Elevated cholesterol is also known to promote microbial infections by HIV, mycobacteria, and malarial parasites. Yet the molecular determinants of pathogens that underlie cholesterol sequestration in the vacuole have been difficult to identify. S. Typhimurium is a gram-negative bacterium that causes significant human disease and is also highly amenable to genetic manipulation. Cholesterol accumulates in the intracellular vacuole and the long-term aim of this proposal is to exploit genetic methods available in S. Typhimurium to identify bacterial determinants that underlie both infection and pathogenesis. The study will contribute to our basic understanding of how S. Typhimurium alters cholesterol distribution in cells and new strategies for limiting bacterial proliferation in the Salmonella-containing vacuoles (SCV). Molecular, genetic tools including gene knock outs and gene screens, combined with high resolution imaging techniques and biochemical subcellular fractionation assays, will be used to identify bacterial genes that are responsible for acquisition and balance of cholesterol in the SCV. The consequence of ablation of bacterial gene products will be evaluated in an in vitro tissue culture model as well as in animals. Their homologues in other microbial pathogens as well as mammalian cells may reveal new pathogenic determinants and new mechanisms underlying cholesterol homeostasis.